Thioredoxin 1 is inactivated due to oxidation induced by peroxiredoxin under oxidative stress and reactivated by the glutaredoxin system

Abstract

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H2O2. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.

Keywords: Hydrogen Peroxide; Oxidative Stress; Peroxiredoxin; Redox Signaling; Thiol; Thioredoxin.

FIGURE 1.

Redox state of Trx1 in A549 cells under oxidative stress. A, principle of redox Western blot analysis. To prepare mobility standards, cell lysates were denatured with urea and fully reduced with DTT. Varying molar ratios of IAA to IAM were incubated with the reduced Trx1 containing five cysteines, producing six protein isoforms with the introduced number of acidic carboxymethylthiol adducts (–SA⁻) and neutral amidomethylthiol adducts (–SM). During urea-PAGE, the ionized –SA⁻ group resulted in faster protein migration toward the anode. Therefore, the six isoforms were separated and used as a mobility standard for representing the number of –SA⁻. To determine the redox state of Trx1 in cells, cells were lysed in urea lysis buffer containing IAM. After the free thiols of Trx1 were alkylated by IAM, cell lysates were precipitated by ice-cold acetone HCl. The precipitate was washed with ice-cold acetone HCl two more times to remove excess IAM. The precipitate was then resuspended in urea lysis buffer containing DTT to reduce the disulfides of Trx1. The free thiols of Trx1 were alkylated by IAA. The alkylated Trx1 in cell lysates was separated according to the charge amount, representing the initial amount of free thiols of Trx1. B, A549 cells were exposed to oxidative stress (15 mm H₂O₂) for the indicated times (lanes 2–5), and the redox state of Trx1 in A549 cells was detected by redox Western blot analysis. Lane 1, artificial mobility standards (M).

FIGURE 2.

Redox state shift of human Trx1-S₂ and Trx1SGPS in the presence of Prx1 and/or H₂O₂. Trx1-S₂ was prepared as described under “Experimental Procedures.” 10 μm Trx1-S₂ or reduced Trx1SGPS was incubated with the indicated concentrations of Prx1/H₂O₂ (lanes 1–5) for 5 min at 37 °C. The redox state of Trx1 and Trx1SGPS was detected by redox urea-PAGE analysis. Lane 6, artificial mobility standards (M).

FIGURE 3.

Redox state of Trx1 in HeLa cells treated with ebselen. HeLa cells were exposed to the indicated concentrations of ebselen for 2 h with or without 0.1 mm BSO pretreatment (lanes 2–7). The redox state of Trx1 in HeLa cells was detected with by redox Western blot analysis. Lane 1, artificial mobility standards (M).

FIGURE 4.

Reduction of oxidized Trx1SGPS and Trx1-S₄ by the GSH/Grx system. A, 45 nm glutathione reductase, 0.25 mm NADPH, and 20 μm oxidized Trx1SGPS were added to cuvettes for the GSH reduction assay in the presence of 3 (solid black line), 6 (dashed line), and 10 (dotted line) mm GSH. 0.25 mm NADPH and 20 μm oxidized Trx1SGPS were added to cuvettes for the TrxR1 reduction assay in the presence of 10 nm TrxR1 (solid gray line). The absorbance change at 340 nm was monitored. B, 45 nm glutathione reductase, 0.25 mm NADPH, 1 mm GSH, and the indicated amounts of oxidized Trx1SGPS/insulin were added to cuvettes for the reduction assay with or without 1 μm human Grx1. The absorbance change at 340 nm was monitored. C, 45 nm glutathione reductase, 0.25 mm NADPH, 1 mm GSH, and the indicated amounts of oxidized WT Trx1 were added to cuvettes for the reduction assay with or without 1 μm human Grx1. The absorbance change at 340 nm was monitored.

FIGURE 5.

Reduction of oxidized Trx1SGPS by the E. coli Grx1 C14S mutant protein. 45 nm glutathione reductase, 0.25 mm NADPH, 1 mm GSH, and 20 (solid black line) or 40 (dashed line) μm oxidized Trx1SGPS were added to cuvettes for the Grx reduction assay in the presence of 1 μm E. coli Grx1 C14S (GrxC14S) mutant protein. 0.25 mm NADPH and 20 μm oxidized Trx1SGPS were added to cuvettes for the TrxR1 reduction assay in the presence of 10 nm TrxR1 (solid gray line). The absorbance change at 340 nm was monitored.

FIGURE 6.

Proposed mechanism of Trx1-S₄ in redox regulation and oxidative stress. A, proposed mechanism of reduction of oxidized Trx1 by GSH/Grx1. The non-active site disulfide is reduced by GSH via a simple chemical reaction. This reaction is stimulated by Grx1 through the monothiol mechanism. B, the oxidative signal is transmitted from H₂O₂ to Trx1-S₄ via Prx1. Trx1-S₄ is inactive and therefore results in Prx1 oxidation and H₂O₂ accumulation, which is involved in redox regulation, including inhibition of protein-tyrosine phosphatases and PTEN. The ASK1, NF-κB, p53, Ref-1, and AP-1 pathways will also benefit from the inactivation of Trx1.